A composite hydrogel loaded with asiaticoside and a preparation method and application thereof

Abstract

The application discloses a kind of water gel of loading asiaticoside, by the Michael addition reaction crosslinking single-network chemical crosslinking hydrogel formed by end acrylate poly-β-amino ester and eight-arm polyethylene glycol thiol, and the water gel is uniformly loaded with therapeutically effective amount of asiaticoside in it;The loading concentration of asiaticoside in the water gel is 0.2-2.0 mg / mL;Preparation method includes dissolving end acrylate poly-β-amino ester in sterile PBS or physiological saline to obtain solution A;Asiaticoside powder is dissolved in solution A;Eight-arm polyethylene glycol thiol is dissolved in sterile PBS to obtain solution B;Solution A is quickly mixed with solution B to obtain asiaticoside-loaded water gel at room temperature or below 4°C;The application solves the problem that the half-life of existing AS preparation is short, and repeated injection is needed, while avoiding the use of complex crosslinking system or potential toxic crosslinking agent.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a composite hydrogel and its preparation method. Background Technology

[0002] Osteoarthritis (OA) is a chronic joint disease characterized by degeneration of articular cartilage, inflammation of the synovium, and pain. Intra-articular injection is a commonly used method of local drug administration, but free drugs (such as hyaluronic acid and hormones) are rapidly cleared from the joint cavity, requiring frequent injections and resulting in poor patient compliance.

[0003] Asiaticoside (AS) is a natural triterpenoid saponin with anti-inflammatory, collagen-promoting, and chondrogenic effects. However, its water solubility is generally low, and its in vivo half-life is short, making it difficult to maintain effective concentrations through direct injection. Injectable hydrogels can serve as drug reservoirs, achieving long-acting sustained release. Poly(β-amino ester) (PBAE) is a biodegradable cationic polymer with acrylate end groups, which can undergo Michael addition reactions with thiol groups to form hydrogels. Eight-arm polyethylene glycol thiol (8-arm PEG-SH) has multiple thiol end groups and can serve as a highly efficient cross-linking agent to form a uniform three-dimensional network. Currently, there are no reports on the use of PBAE and eight-arm PEG-SH composites loaded with AS for intra-articular osteoarthritis (OA) treatment. Summary of the Invention

[0004] The purpose of this invention is to provide an injectable, biodegradable, sustained-release asiaticoside PBAE / 8-arm PEG-SH hydrogel and its preparation method, which solves the problems of short half-life and repeated injection required by existing AS formulations, while avoiding the use of complex cross-linking systems or potentially toxic cross-linking agents.

[0005] To achieve the above objectives, the following technical solution is adopted: A hydrogel loaded with asiaticoside is formed by cross-linking terminal acrylated polyβ-amino ester with eight-arm polyethylene glycol thiol groups through Michael addition reaction to form a single-network chemical cross-linked hydrogel, and the hydrogel is uniformly loaded with a therapeutically effective amount of asiaticoside.

[0006] According to the above scheme, the loading concentration of asiaticoside in the hydrogel is 0.2 to 2.0 mg / mL, preferably 1.0 mg / mL.

[0007] This invention also provides a method for preparing the above-mentioned hydrogel loaded with asiaticoside, comprising the following steps: (1) Dissolve the acrylate-terminated polyβ-amino ester in sterile PBS or physiological saline to obtain solution A; (2) Dissolve the asiaticoside powder in solution A; (3) Dissolve the eight-arm polyethylene glycol thiol group in sterile PBS to obtain solution B; (4) At room temperature or below 4°C, solution A and solution B are rapidly mixed to obtain a hydrogel loaded with asiaticoside.

[0008] According to the above scheme, the preparation method of the acrylate-terminated polyβ-amino ester includes the following steps: Polyethylene glycol diacrylate and cystamine dihydrochloride were dissolved in DMSO at a molar ratio of 2.5:1. The pH was adjusted to 8.0, and the mixture was stirred at 60°C for 8 hours. The resulting product was purified to obtain acrylate-terminated polyβ-amino ester.

[0009] According to the above scheme, the concentration of solution A in step (1) is 8 to 15 mg / mL.

[0010] According to the above scheme, the concentration of asiaticoside in step (2) is 0.2 to 2.0 mg / mL.

[0011] According to the above scheme, the molecular weight of the eight-arm polyethylene glycol thiol in step (3) is 10k-40k and the thiol functionality is 6-10; in the preferred scheme, the molecular weight is selected as 10k, 20k or 40k and the thiol functionality is 8.

[0012] According to the above scheme, the volume ratio of solution A to solution B in step (4) is 1:1.

[0013] According to the above scheme, in step (4), the molar ratio of thiol group carried by the eight-arm polyethylene glycol thiol group to acrylate group carried by the terminally acrylated polyβ-amino ester is 1:(0.5-3). In the optimized scheme, the molar ratio of thiol group to acrylate group is 1:1.

[0014] The present invention also provides the application of the above-mentioned hydrogel loaded with asiaticoside in injectable therapeutic drugs for osteoarthritis.

[0015] The disulfide bonds in the PBAE backbone are sensitive to elevated glutathione (GSH) levels in the inflammatory joint microenvironment, enabling responsive degradation and thus controlling the release rate of arthritis atherosclerosis (AS). 8-arm PEG-SH provides high cross-linking density, enhancing the mechanical strength of the hydrogel and adapting to shear and compressive stresses within the joint cavity. The released AS exerts anti-inflammatory and chondroprotective effects by inhibiting the NF-κB pathway and reducing inflammatory factors (IL-1β, TNF-α).

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: Injectable and in-situ gels: The precursor solution has low viscosity and can be injected through a 26G-30G fine needle, avoiding open surgery and making the operation simple.

[0017] Biodegradability: PBAE degradation products are oligomers, glycols and cystamine analogs. PEG is an FDA-approved material and its degradation products are non-toxic.

[0018] Adjustable sustained-release properties: By changing the PBAE concentration, PEG-SH molecular weight, mercapto / acrylate ratio, and AS drug loading, the drug release rate can be controlled from several days to several weeks.

[0019] Mechanical property adaptation: The eight-arm PEG-SH forms a uniform highly cross-linked network, and the hydrogel compression modulus can reach 30-80 kPa, which is suitable for the joint cavity environment.

[0020] The preparation process is simple: no photoinitiator, enzyme or other catalyst is required, thus avoiding potential toxicity or immunogenicity. Attached Figure Description

[0021] Figure 1 Scanning electron microscope image of the hydrogel obtained in Example 1.

[0022] Figure 2 Gelation time curves for different mercapto / acrylate molar ratios.

[0023] Figure 3 Cumulative release curves of AS in vitro at different GSH concentrations.

[0024] Figure 4 Results of Safranin O-Fix Green staining and OARSI score after intra-articular injection in a DMM osteoarthritis model. Detailed Implementation

[0025] The following embodiments further illustrate the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0026] A specific embodiment provides an end-acrylated polyβ-amino ester: Polyethylene glycol diacrylate (PEGDA, Mn=700) and cystamine dihydrochloride were dissolved in DMSO at a molar ratio of 2.5:1, and the pH was adjusted to 8.0 with an alkaline reagent. The reaction was carried out at 60°C and 600 rpm for 8 hours to obtain a PBAE prepolymer with acrylate end groups (containing disulfide bonds that can be reduced and cleaved by glutathione). After the reaction was completed, the prepolymer was extracted five times with ice-cold diethyl ether to remove unreacted monomers, and then vacuum dried to obtain purified PBAE.

[0027] The specific embodiment uses 8-arm polyethylene glycol thioglycolate (8-arm PEG-SH) (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number P968649, Mw=10000).

[0028] The specific implementation method uses asiaticoside powder (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number A964372, purity 98%).

[0029] Unless otherwise specified, all other raw materials used in the specific implementation method are obtained through commercial purchase.

[0030] Example 1: Preparation of basic formulation hydrogel PBAE (PEGDA700:cystamine dihydrochloride = 2.5:1) was synthesized and stored in a dry place.

[0031] Weigh 100 mg PBAE and dissolve it in 1 mL PBS (10% w / v). Add 1 mg AS and sonicate to dissolve, to obtain solution A (AS concentration 1 mg / mL).

[0032] Weigh out an eight-arm PEG-SH (molecular weight 20k, thiol content approximately 0.4 mmol / g) and dissolve it in PBS to prepare a solution with a concentration equal to the number of moles of thiol in PBAE. Calculation: The number of moles of acrylate in 1 mL of a 10% PBAE solution = (100 mg / average molecular weight PBAE) × 2 (each PBAE chain has 2 end groups). Estimation of the average molecular weight of PBAE: PEGDA700 copolymerized with cystamine dihydrochloride (molecular weight approximately 225), approximately Mn~2000, therefore 1 mL of a 10% solution contains 0.05 mmol of PBAE and 0.10 mmol of acrylate. The eight-arm PEG-SH 20k requires 0.10 mmol of thiol, which is approximately (0.10 / 8) × 20000 = 250 mg, dissolved in 1 mL of PBS to obtain solution B.

[0033] Equal volumes of solution A and solution B were mixed and quickly drawn into a syringe, then injected into a mold at 37°C. A gel formed in approximately 8–12 minutes. A scanning electron microscope image of the hydrogel obtained in this example is attached. Figure 1 As shown.

[0034] Example 2: Optimization of gelation time With a fixed PBAE concentration of 10% (w / v), the amount of PEG-SH in the eight arms was adjusted to make the molar ratio of mercapto to acrylate 0.8:1, 1:1, and 1.2:1, respectively.

[0035] Gelation time curves for different mercapto / acrylate molar ratios are shown in the appendix. Figure 2 As shown. At a 1:1 ratio, the gelation time is approximately 10 minutes, providing ample operating window; at 1.2:1, the gelation time is shortened to 5 minutes, with a slight increase in mechanical strength; at 0.8:1, the gelation time exceeds 20 minutes, and gelation is incomplete. A ratio of 1:1 to 1.2:1 is preferred.

[0036] Example 3: Different AS drug loading Following the method in Example 1, the concentration of PBAE was fixed at 10%, the number of equimolar thiol groups in the eight-arm PEG-SH was set to 0.2, 0.5, 1.0, and 2.0 mg / mL, respectively.

[0037] The in vitro cumulative release curves of AS at different GSH concentrations are attached. Figure 3 As shown. In vitro release (pH 7.4, containing 10 mM GSH): release period is approximately 14–21 days; the higher the drug loading, the greater the cumulative release, with an initial burst release of <20%. 1.0 mg / mL is preferred as the therapeutic concentration.

[0038] Example 4: Pharmacological evaluation of a DMM osteoarthritis model Animal model: An OA model was established by surgically severing the medial meniscus ligament of the right knee (DMM method) in 8-week-old C57BL / 6 mice.

[0039] Grouping: On postoperative day 3, intra-articular injections were administered of: (a) PBS control group; (b) blank hydrogel (without AS); (c) AS-loaded hydrogel (AS 1 mg / mL, prepared according to Example 1). Ten animals were in each group, and each animal received 20 μL.

[0040] Assessment: The patient was euthanized 8 weeks post-surgery, and the knee joint was harvested for micro-CT, Safranin O-Fix Green staining of tissue sections, and OARSI scoring.

[0041] The results of intra-articular injection of Safranin O-Fix Green staining and OARSI score in the DMM osteoarthritis model are attached. Figure 4 As shown in the figure, cartilage degeneration was significantly reduced in the treatment group, and the OARSI score (2.1±0.6) was significantly lower than that in the PBS group (5.8±0.9) and the blank gel group (5.2±1.0). The synovial inflammation score was also significantly reduced. The AS concentration of the joint lavage fluid showed that it was still at a therapeutic level on day 14.

[0042] Example 5: Molecular weight regulation of PBAE Different molecular weight PBAEs were synthesized by adjusting the molar ratio of PEGDA700 to cystamine dihydrochloride (2:1, 2.5:1, 3:1). Higher ratios resulted in larger molecular weights. The PBAE obtained at a ratio of 2.5:1 exhibited the best gelation time and mechanical properties. At a ratio of 3:1, the reaction was incomplete, leading to increased free acrylate and heterogeneous gelation.

Claims

1. A hydrogel loaded with asiaticoside, characterized in that... A single-network chemically cross-linked hydrogel is formed by cross-linking terminal acrylated polyβ-amino esters with eight-arm polyethylene glycol thiol groups via Michael addition reaction, and the hydrogel is uniformly loaded with a therapeutically effective amount of asiaticoside.

2. The hydrogel loaded with asiaticoside as described in claim 1, characterized in that... The loading concentration of asiaticoside in the hydrogel is 0.2–2.0 mg / mL.

3. The method for preparing the hydrogel loaded with asiaticoside according to claim 1, characterized in that... Includes the following steps: (1) Dissolve the acrylate-terminated polyβ-amino ester in sterile PBS or physiological saline to obtain solution A; (2) Dissolve the asiaticoside powder in solution A; (3) Dissolve the eight-arm polyethylene glycol thiol group in sterile PBS to obtain solution B; (4) At room temperature or below 4°C, solution A and solution B are rapidly mixed to obtain a hydrogel loaded with asiaticoside.

4. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... The method for preparing the acrylate-terminated polyβ-amino ester includes the following steps: Polyethylene glycol diacrylate and cystamine dihydrochloride were dissolved in DMSO at a molar ratio of 2.5:

1. The pH was adjusted to 8.0, and the mixture was stirred at 60°C for 8 hours. The resulting product was purified to obtain acrylate-terminated polyβ-amino ester.

5. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... The concentration of solution A in step (1) is 8–15 mg / mL.

6. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... In step (2), the concentration of asiaticoside is 0.2–2.0 mg / mL.

7. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... The eight-arm polyethylene glycol thiol in step (3) has a molecular weight of 10k-40k and a thiol functionality of 6-10.

8. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... In step (4), the volume ratio of solution A to solution B is 1:

1.

9. The method for preparing the hydrogel loaded with asiaticoside as described in claim 3, characterized in that... In step (4), the molar ratio of thiol group carried by the eight-arm polyethylene glycol thiol group to acrylate group carried by the terminal acrylated polyβ-amino ester is 1:(0.5-3).

10. The use of the hydrogel loaded with asiaticoside according to claim 1 in an injectable therapeutic agent for osteoarthritis.

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